Ubiquitin-dependent remodeling of the actin cytoskeleton drives cell fusion

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Ligand-specific dynamics of the progesterone receptor in living cells and during chromatin remodeling in vitro

Progesterone receptor (PR), a member of the nuclear receptor superfamily, is a key regulator of several processes in reproductive function. We have studied the dynamics of the interaction of PR with a natural target promoter in living cells through the use of fluorescence recovery after photobleaching (FRAP) analysis and also have characterized the dynamics of the interaction of PR with the mouse mammary tumor virus (MMTV) promoter reconstituted into chromatin in vitro. In photobleaching experiments, PR in the presence of the agonist R5020 exhibits rapid exchange with the MMTV promoter in living cells. Two PR antagonists, RU486 and ZK98299, have opposite effects on receptor dynamics in vivo. In the presence of RU486, PR binds to the promoter and is exchanged more slowly than the agonist-activated receptor. In contrast, PR bound to ZK98299 is not localized to the promoter and exhibits higher mobility in the nucleoplasm than the agonist-bound receptor. Significantly, PR bound to R5020 or RU486 can recruit the SWI/SNF chromatin remodeling complex to the promoter, but PR activated with ZK98299 cannot. Furthermore, we found ligand-specific active displacement of PR from the MMTV promoter during chromatin remodeling in vitro and conclude that the interaction of PR with chromatin is highly dynamic both in vivo and in vitro. We propose that factor displacement during chromatin remodeling is an important component of receptor mobility and that ligand-specific interactions with remodeling complexes can strongly influence receptor nuclear dynamics and rates of exchange with chromatin in living cells.     

Tumor cell pseudopodial protrusions. Localized signaling domains coordinating cytoskeleton remodeling, cell adhesion, glycolysis, RNA translocation, and protein translation

The pseudopodial protrusions of Moloney sarcoma virus (MSV)-Madin-Darby canine kidney (MDCK)-invasive (INV) variant cells were purified on 1-microm pore polycarbonate filters that selectively allow passage of the pseudopodial domains but not the cell body. The purified pseudopodial fraction contains phosphotyrosinated proteins, including Met and FAK, and various signaling proteins, including Raf1, MEK1, ERK2, PKBalpha (Akt1), GSK3alpha, GSK3beta, Rb, and Stat3. Pseudopodial proteins identified by liquid chromatography tandem mass spectrometry included actin and actin-regulatory proteins (ERM, calpain, filamin, myosin, Sra-1, and IQGAP1), tubulin, vimentin, adhesion proteins (vinculin, talin, and beta1 integrin), glycolytic enzymes, proteins associated with protein translation, RNA translocation, and ubiquitin-mediated protein degradation, as well as protein chaperones (HSP90 and HSC70) and signaling proteins (RhoGDI and ROCK). Inhibitors of MEK1 (U0126) and HSP90 (geldanamycin) significantly reduced MSV-MDCK-INV cell motility and pseudopod expression, and geldanamycin treatment inhibited Met phosphorylation and induced the expression of actin stress fibers. ROCK inhibition did not inhibit cell motility but transformed the pseudopodial protrusions of MSV-MDCK-INV cells into extended lamellipodia. Dominant negative Rho disrupted pseudopod expression and, in serum-starved cells, L-alpha-lysophosphatidic acid (oleoyl) activation of Rho induced pseudopodial protrusions or, in the presence of the ROCK inhibitor, extended lamellipodia. RNA was localized to the actin-rich pseudopodial domains of MSV-MDCK-INV cells, but the extent of colocalization with dense actin ruffles was reduced in the extended lamellipodia formed upon ROCK inhibition. Rho/ROCK activation in epithelial tumor cells therefore regulates RNA translocation to a pseudopodial domain that contains proteins involved in signaling, cytoskeleton remodeling, cell adhesion, glycolysis, and protein translation and degradation.     

The spectrin cytoskeleton is crucial for adherent and invasive bacterial pathogenesis

Various enteric bacterial pathogens target the host cell cytoskeletal machinery as a crucial event in their pathogenesis. Despite thorough studies detailing strategies microbes use to exploit these components of the host cell, the role of the spectrin-based cytoskeleton has been largely overlooked. Here we show that the spectrin cytoskeleton is a host system that is hijacked by adherent (Entropathogenic Escherichia coli [EPEC]), invasive triggering (Salmonella enterica serovar Typhimurium [S. Typhimurium]) and invasive zippering (Listeria monocytogenes) bacteria. We demonstrate that spectrin cytoskeletal proteins are recruited to EPEC pedestals, S. Typhimurium membrane ruffles and Salmonella containing vacuoles (SCVs), as well as sites of invasion and comet tail initiation by L. monocytogenes. Spectrin was often seen co-localizing with actin filaments at the cell periphery, however a disconnect between the actin and spectrin cytoskeletons was also observed. During infections with S. Typhimurium ΔsipA, actin-rich membrane ruffles at characteristic sites of bacterial invasion often occurred in the absence of spectrin cytoskeletal proteins. Additionally, early in the formation of L. monocytogenes comet tails, spectrin cytoskeletal elements were recruited to the surface of the internalized bacteria independent of actin filaments. Further studies revealed the presence of the spectrin cytoskeleton during SCV and Listeria comet tail formation, highlighting novel cytoplasmic roles for the spectrin cytoskeleton. SiRNA targeted against spectrin and the spectrin-associated proteins severely diminished EPEC pedestal formation as well as S. Typhimurium and L. monocytogenes invasion. Ultimately, these findings identify the spectrin cytoskeleton as a ubiquitous target of enteric bacterial pathogens and indicate that this cytoskeletal system is critical for these infections to progress.     

Unconventional myosins at the crossroad of signal transduction and cytoskeleton remodeling

The cytoplasm of eukaryotic cells is a complex milieu and unraveling how its unique cytoarchitecture is achieved and maintained is a central theme in modern cell biology. The actin cytoskeleton is essential for the maintenance of cell shape and locomotion, and also provides tracks for active intracellular transport. Myosins, the actin-dependent motor proteins form a superfamily of at least 15 structural classes and have been identified in a wide variety of organisms, making the presence of actin and myosins a hallmark feature of eukaryotes. Direct connections of myosins to a variety of cellular tasks are now emerging, such as in cytokinesis, phagocytosis, endocytosis, polarized secretion and exocytosis, axonal transport. Recent studies reveal that myosins also play an essential role in many aspects of signal transduction and neurosensation.     

Cell adhesion dynamics and actin cytoskeleton reorganization in HepG2 cell aggregates

The temporal dependence of cytoskeletal remodelling on cell-cell contact in HepG2 cells has been established here. Cell-cell contact occurred in an ultrasound standing wave trap designed to form and levitate a 2-D cell aggregate, allowing intercellular adhesive interactions to proceed, free from the influences of solid substrata. Membrane spreading at the point of contact and change in cell circularity reached 50% of their final values within 2.2 min of contact. Junctional F-actin increased at the interface but lagged behind membrane spreading, reaching 50% of its final value in 4.4 min. Aggregates had good mechanical stability after 15 min in the trap. The implication of this temporal dependence on the sequential progress of adhesion processes is discussed. These results provide insight into how biomimetic cell aggregates with some liver cell functions might be assembled in a systematic, controlled manner in a 3-D ultrasound trap.     

Unconventional myosins at the crossroad of signal transduction and cytoskeleton remodeling

Summary The cytoplasm of eukaryotic cells is a complex milieu and unraveling how its unique cytoarchitecture is achieved and maintained is a central theme in modern cell biology. The actin cytoskeleton is essential for the maintenance of cell shape and locomotion, and also provides tracks for active intracellular transport. Myosins, the actin-dependent motor proteins form a superfamily of at least 15 structural classes and have been identified in a wide variety of organisms, making the presence of actin and myosins a hallmark feature of eukaryotes. Direct connections of myosins to a variety of cellular tasks are now emerging, such as in cytokinesis, phagocytosis, endocytosis, polarized secretion and exocytosis, axonal transport. Recent studies reveal that myosins also play an essential role in many aspects of signal transduction and neurosensation.     

Skin and bones: the bacterial cytoskeleton, cell wall, and cell morphogenesis

The bacterial world is full of varying cell shapes and sizes, and individual species perpetuate a defined morphology generation after generation. We review recent findings and ideas about how bacteria use the cytoskeleton and other strategies to regulate cell growth in time and space to produce different shapes and sizes.     

Calcium,protease activation,and cytoskeleton remodeling underlie growth cone formation and neuronal regeneration

The cytoarchitecture, synaptic connectivity, and physiological properties of neurons are determined during their development by the interactions between the intrinsic properties of the neurons and signals provided by the microenvironment through which they grow. Many of these interactions are mediated and translated to specific growth patterns and connectivity by specialized compartments at the tips of the extending neurites: the growth cones (GCs). The mechanisms underlying GC formation at a specific time and location during development, regeneration, and some forms of learning processes, are therefore the subject of intense investigation. Using cultured Aplysia neurons we studied the cellular mechanisms that lead to the transformation of a differentiated axonal segment into a motile GC. We found that localized and transient elevation of the free intracellular calcium concentration ([Ca²⁺] _i ) to 200–300 M induces GC formation in the form of a large lamellipodium that branches up into growing neurites. By using simultaneous on-line imaging of [Ca²⁺] _i and of intraaxonal proteolyticactivity, we found that the elevated [Ca²⁺] _i activate proteases in the region in which a GC is formed. Inhibition of the calcium-activated proteases prior to the local elevation of the [Ca²⁺] _i blocks the formation of GCs. Using retrospective immunofluorescent methods we imaged the proteolysis of the submembrane spectrin network, and the restructuring of the cytoskeleton at the site of GC formation. The restructuring of the actin and microtubule network leads to local accumulation of transported vesicles, which then fuse with the plasma membrane in support of the GC expansion.     

Multi-patterned dynamics of mitochondrial fission and fusion in a living cell

Mitochondria are highly-dynamic organelles, but it is challenging to monitor quantitatively their dynamics in a living cell. Here we developed a novel approach to determine the global occurrence of mitochondrial fission and fusion events in living human epithelial cells (Hela) and mouse embryonic fibroblast cells (MEF). Distinct patterns of sequential events including fusion followed by fission (Fu-Fi), the so-called "kiss and run" model previously described, fission followed by fusion (Fi-Fu), fusion followed by fusion (Fu-Fu), and fission followed by fission (Fi-Fi) were observed concurrently. The paired events appeared in high frequencies with short lifetimes and large sizes of individual mitochondria, as compared to those for unpaired events. The high frequencies of paired events were found to be biologically significant. The presence of membrane uncoupler CCCP enhanced the frequency of paired events (from both Fu-Fi and Fi-Fu patterns) with a reduced mitochondrial size. Knock-out of mitofusin protein Mfn1 increased the frequency of fission with increased lifetime of unpaired events whereas deletion of both Mfn1 and Mfn2 resulted in an instable dynamics. These results indicated that the paired events were dominant but unpaired events were not negligible, which provided a new insight into mitochondrial dynamics. In addition to kiss and run model of action, our data suggest that, from a global visualization over an entire cell, multiple patterns of action appeared in mitochondrial fusion and fission.     

Inter-regulatory dynamics of phospholipase D and the actin cytoskeleton

Phospholipase D activity has been extensively implicated in the regulation of the actin cytoskeleton. Through this regulation the enzyme controls a number of physiological functions such as cell migration and adhesion and, it also is implicated in the regulation of membrane trafficking. The two phospholipase Ds are closely implicated with the control of the ARF and Rho families of small GTPases. In this article it is proposed that PLD2 plays the role of ‘master regulator’ and in an ill-defined manner regulates Rho function, PLD1 activity is downstream of this activation, however the generated phosphatidic acid controls changes in cytoskeletal organisation through its regulation of phosphatidylinositol-4-phosphate-5-kinase activity.     

Biphasic effect of danazol on human vascular endothelial cell permeability and f-actin cytoskeleton dynamics

Breakdown of endothelial barrier function is a hallmark event across a variety of pathologies such as inflammation, cancer, and diabetes. It has also been appreciated that steroid hormones impart direct biological activity on endothelial cells at many levels. The purpose of this investigation was to explore the effect of the androgen-like steroid, danazol, on endothelial cell barrier function in vitro. Primary human endothelial cells exposed to 0.01-50 μM danazol were evaluated for changes in permeability. We found that danazol altered endothelial permeability in a biphasic manner in which nanomolar concentrations enhance barrier function while micromolar concentrations are detrimental. Monitoring of trans-endothelial electrical resistance demonstrated that these barrier enhancing effects were rapid (within 5 min) and lasted for over 24h. Analysis of intracellular f-actin organization showed that barrier enhancement also correlated with the formation of a submembranous cortical actin ring. Conversely, at higher danazol concentrations, contractile cell phenotypes were observed, represented by stress fiber formation. Competitive binding studies performed using steroid hormone receptor antagonists proved that this activity is the result of androgen and estrogen receptor ligation. These findings suggest that low dose danazol may provide a therapeutic window for diseases involving vascular leakage.     

Modes of remodeling in the cortical cytoskeleton of vascular endothelial cells

The cortical cytoskeleton of vascular endothelial cells plays an important role in responding to mechanical stimuli and controlling the distribution of cell surface proteins. Here, we have used atomic force microscopy to visualize the dynamics of cortical cytoskeleton in living bovine pulmonary artery endothelial cells. We demonstrate that the cortical cytoskeleton, organized as a complex polygonal mesh, is highly dynamic and shows two modes of remodeling: intact-boundary-mode where mesh element boundaries remain intact but move at approximately 0.08 microm/min allowing the mesh element to change shape, and altered-boundary-mode where new mesh boundaries form and existing ones disappear.     

Coronin-1A links cytoskeleton dynamics to TCR alpha beta-induced cell signaling

Actin polymerization plays a critical role in activated T lymphocytes both in regulating T cell receptor (TCR)-induced immunological synapse (IS) formation and signaling. Using gene targeting, we demonstrate that the hematopoietic specific, actin- and Arp2/3 complex-binding protein coronin-1A contributes to both processes. Coronin-1A-deficient mice specifically showed alterations in terminal development and the survival of alpha beta T cells, together with defects in cell activation and cytokine production following TCR triggering. The mutant T cells further displayed excessive accumulation yet reduced dynamics of F-actin and the WASP-Arp2/3 machinery at the IS, correlating with extended cell-cell contact. Cell signaling was also affected with the basal activation of the stress kinases sAPK/JNK1/2; and deficits in TCR-induced Ca2+ influx and phosphorylation and degradation of the inhibitor of NF-kappaB (I kappa B). Coronin-1A therefore links cytoskeleton plasticity with the functioning of discrete TCR signaling components. This function may be required to adjust TCR responses to selecting ligands accounting in part for the homeostasis defect that impacts alpha beta T cells in coronin-1A deficient mice, with the exclusion of other lympho/hematopoietic lineages.     

Analysis of the signaling pathways regulating Src-dependent remodeling of the actin cytoskeleton

Cell adhesion to the extracellular matrix is mediated by adhesion receptors, mainly integrins, which upon interaction with the extracellular matrix, bind to the actin cytoskeleton via their cytoplasmic domains. This association is mediated by a variety of scaffold and signaling proteins, which control the mechanical and signaling activities of the adhesion site. Upon transformation of fibroblasts with active forms of Src (e.g., v-Src), focal adhesions are disrupted, and transformed into dot-like contacts known as podosomes, and consisting of a central actin core surrounded by an adhesion ring. To clarify the mechanism underlying Src-dependent modulation of the adhesive phenotype, and its influence on podosome organization, we screened for the effect of siRNA-mediated knockdown of tyrosine kinases, MAP kinases and phosphatases on the reorganization of the adhesion-cytoskeleton complex, induced by a constitutively active Src mutant (SrcY527F). In this screen, we discovered several genes that are involved in Src-induced remodeling of the actin cytoskeleton. We further showed that knockdown of Src in osteoclasts abolishes the formation of the podosome-based rings and impairs cell spreading, without inducing stress fiber development. Our work points to several genes that are involved in this process, and sheds new light on the molecular plasticity of integrin adhesions.     

Heterologous expression of acetylcholinesterase affects proliferation and glial cytoskeleton of adherent chicken retinal cells

Besides its function at cholinergic synapses, acetylcholinesterase (AChE) exerts structural functions on neural differentiation, independent of its enzymatic activity. To elucidate such functions, we have previously heterologously expressed AChE in histotypic retinal reaggregates, revealing strong effects on their histogenesis, particularly on Müller glia processes. To further resolve these findings at a less complex cellular level, in this study we transfected adherent retinal cells of the chick embryo after 2 days i.c. with a sense pSVK3-AChErab-cDNA expression vector encoding for the entire rabbit AChE gene by calcium phosphate precipitation. Northern blots using digoxigenin (DIG)-labeled rabbit cDNA revealed a pronounced level of rabbit AChE mRNA in AChE-transfected cells. Western blot analysis established an increase in the endogenous AChE protein in transfected cells. Noticeably, AChE activity was not much affected, indicating a post-translational regulation of overall AChE activity. As a corollary, 5'-bromo-2'-deoxyuridine (BrdU) studies showed a decrease in cell proliferation. Exploring changes of the Müller glia, the cytoskeletal protein vimentin was found to be increased in transfected cells. Vimentin-stained processes are longer, thicker and more orderly arranged. In conclusion, exogenous expression of rabbit AChE in chicken retinal monolayers exerts a structural function on glial cytoskeletal organization, independent of AChE activity.